From our previous studies we determined that activated rat platelets induce cholesterol accumulation in cultured rat smooth muscle cells and human fibroblasts, whereas, activated human platelets do not. We would like to establish the mechanism of platelet-mediated cellular cholesterol accumulation and the basis for the differential response with human and rat platelets. In this study we have examined changes in platelet phospholipids induced by activation to determine if this could be a factor in platelet-mediated cholesterol accumulation. We have observed that activation of rat platelets results in significant phospholipid hydrolysis (80% of initial values). This results in an increased cholesterol to phospholipid molar ratio and could potentially promote redistribution of cholesterol from the platelets to other cells or blood lipoproteins. The change in phospholipid content observed in activated rat platelets may represent a more generalized mechanism to promote cholesterol removal from dying cells. When rat platelets were activated they released significant lactate dehydrogenase suggesting that the platelets were dying. By contrast, when human platelets were activated they showed no significant change in phospholipid content or C/P ratio nor did they release nearly as much lactate dehydrogenase as rat platelets. This suggests that the human platelets remained viable after activation. Further studies of platelet-mediated cellular cholesterol accumulation should help elucidate the mechanism(s) of cholesterol removal from cells that die in atherosclerotic lesions and thrombi associated with atherosclerotic lesions. The cholesterol burden from dying cells may contribute significantly to the overall accumulation of cholesterol within atherosclerotic lesions.